HTRF Human Ki-67 Detection Kit, 500 Assay Points

Ki-67 protein expression on starved cultured cells upon stimulation/re-feeding with 24h 10% FCS

A serum starvation and refeeding process was used to imitate the cell cycle using human epithelial A549 cell line derived from a lung carcinoma tissue. First, Ham's F-12 medium without FCS (supplemented with 0.5% BSA, culture grade) was used to incubate A549 cells for 72 h to synchronize them (60K or 120K cells per well in 100µL medium.). The medium was then changed to complete medium in six wells (10% FCS), keeping six wells for starved control.On the first microplate, the medium was removed. Cells were then lysed in 50 µL 1X Lysis buffer and 4 µL lysate were transfered to a 384 well white microplate. 12 µL 1X lysis buffer were added, following the Ki-67 HTRF protocol.The same experiment was run in parallel on the second microplate to compare with a brdU incorporation assay, using a commercial BrdU-ELISA kit. Briefly, BrdU was added (10µL/well) after 2h incubation. Medium was removed and cells were fixed and after blocking (1% BSA 30 min). An anti-BrdU peroxidase conjugate was added (90 min incubation RT), followed by the washing and coloration steps.Ki-67 expression increased in cell lysate upon stimulation with FCS compared to the starved cell control. In parallel, BrdU incorporation increased in FCS stimulated cells. The Ki-67 expression correlates with the BrdU incorporation assay. It should be noted that the assay window was larger in the Ki-67 HTRF assay compared to BrdU assay.

 

 

Differential Ki-67 protein expression in confluent and non-confluent A549 tumor cells

​A549 cells were cultured in two T175 flasks in Ham's F-12 complete medium (10% FCS), using two seeding concentrations to obtain either confluent cells or non-confluent cells. The adherent cells were washed with PBS, then dissociated (5 mL cell dissociation buffer), scraped with a rubber policeman and transferred to three 1.5 mL microtubes (10.E6 cells/tube) as technical replicates. After centrifugation (5 min, 400 RCF) the cell pellets were lysed with 500 µL of lysis buffer #4 (30 min RT vortexing). Lysates were diluted 20-fold in 1X lysis buffer and 16 µL of diluted lysate were transferred (triplicates) to a 384 small volume white microtiter plate for Ki-67 HTRF assay. The Ki-67 expression is clearly much higher in non-confluent A549 cells compared to confluent-cells.

 

Differential Ki-67 protein expression in confluent and non-confluent A549 tumor cells

​A549 cells were cultured in two T175 flasks in Ham's F-12 complete medium (10% FCS), using two seeding concentrations to obtain either confluent cells or non-confluent cells. The adherent cells were washed with PBS, then dissociated (5 mL cell dissociation buffer), scraped with a rubber policeman and transferred to three 1.5 mL microtubes (106 cells/tube) as technical replicates. After centrifugation (5 min, 400 RCF) the cell pellets were lysed with 500 µL of lysis buffer #4 (30 min RT vortexing). Lysates were diluted 20-fold in 1X lysis buffer and 16 µL of diluted lysate were transferred (triplicates) to a 384 small volume white microtiter plate for Ki-67 HTRF assay.The Ki-67 expression is clearly much higher in non-confluent A549 cells compared to confluent-cells.